In liquid chromatography it is sometimes found that not all the analytes present in a solution sample are eluted from a chromatographic column. This can be especially problematic in the analysis of peptides using reverse-phase chromatography, where the recovery in particular of hydrophobic peptides known to be present in a sample is often found to be incomplete. The term “chromatography column” will be used herein to denote a flow-through device generally cylindrical in shape having a solid phase separation medium therein. That term is used with respect to columns and cartridges. The solid phase separation media may be particulate beads, fibers or monolithic. It has been found that if the sample solution comprises peptides dissolved in a solvent comprising a relatively high proportion of organic solvent (that is, one comprising a low proportion of an aqueous solvent), recovery is greater, but especially in the case of nanoflow chromatography the amount of organic solvent that can be used to dissolve the sample is frequently limited by the detrimental effect that a relatively large amount of a strong solvent may have, especially when analytes have to be trapped.
In multi-dimensional liquid chromatography, similar problems may be encountered due to the incompatibility of the mobile phase used to elute analytes from the first dimension separation media with the mobile phase requirements of the second dimension separation media.
The following two prior techniques of multi-dimensional chromatography are especially relevant to this invention. The first technique uses a strong cation exchange (SCX) column in the first dimension and a C18 reverse phase (RP) column in the second dimension. The second technique uses two reverse-phase columns operated with different solvents to provide different separations on each column. The first technique was described in 1999 and many variations are described in subsequent publications. The earliest version involved the use of a biphasic column comprising two sections, one section comprising an SCX stationary phase and the other comprising a RP stationary phase. In use, a mixture of peptides was first trapped on the SCX portion and subsequently, a series of fractions each comprising a number of peptides, were released from the SCX section to the RP section by injection of a series of “salt plugs” of gradually increasing concentration. Each fraction released underwent separation on the RP section before the next salt plug was injected. Peptides eluted from the RP section were characterised by electrospray mass spectrometry.
More recently, the second technique in which the SCX column is replaced by a second RP column has gained popularity. In such multi-dimensional RP/RP systems a partial separation may be carried out on the first RP column using a basic mobile phase (for example pH 10.0) and the subsequent further separation on the second mobile phase may be carried out using an acidic mobile phase (for example, pH 2.6).
These multi-dimensional separations have been automated and improved by several research groups. Typically, at least the second RP column is a nanoflow column that provides optimum separation at a flow rate of less than μl/minute, and usually at much lower flow rates. To facilitate injection of a large volume sample and to allow desalting of the fractions prior to the second stage separation, a trap column is sometimes provided between the SCX or first RP column and the second RP column.
A limitation on the usefulness of these two-dimensional separation systems, especially the RP/RP systems where different mobile phases must be used for each stage to provide different types of separation, is the potential incompatibility of the mobile phase used for the first stage separation with that required for the second stage separation. Limitations may also be imposed on the nature of the solvent used to dissolve a sample even with a single-dimensional separation, especially with nanoflow chromatography where the injected sample volume may be large in comparison with the volume of elution solvent used for a separation. A variety of prior techniques for mitigating these problems have been described, usually involving the intermediate trapping of samples either on a separate trap column or on a column used in a multidimensional separation, or by modifying the solvents used for the elution. However, these are usually of limited application and few, if any, are suitable for use with nanoflow columns.